When large numbers of polymer beads, such as more than 10.000 beads, are to be analysed one by one at an acceptable total analysis time, the beads are typically dispersed in a liquid and passed through a measuring section of the analysis instrument.
In order for the beads to be lined up in a flow channel and passed through a measuring section one by one, while at the same time avoiding that beads stick together or to the walls of the flow system, a high flow velocity is required. Typical flow velocities are in the order of 1 m/s (meter per second) is required. Consequently the residence time of each bead in the measuring section is very short, typically less than 1 millisecond. This poses a number of serious problem as not all measuring or analysing devices can operate within such a short “window of opportunity” for measuring and analysing bead properties. Furthermore, the devices which can in fact operate in the “window of opportunity” of less than 1 millisecond often does not work reliably or under optimal conditions with such a short residence time of the beads.
Also, the demands on the measuring components are very high and prevents the application of certain advanced and time consuming measuring methods. If there is a need for sorting the beads on the basis of the measured properties, there is often not enough time to complete the algebra and very complicated mathematical calculations required to establish whether a bead is to be selected or discarded on the basis of the measurement and analysis performed. One reason for this shortcoming of prior art devices for bead sorting is that the sorting unit is typically placed at a very short distance downstream from the measuring section. Accordingly, time consuming mathematical calculations cannot be used in this context as a bead would have passed the sorting unit long before the result of the measuring of bead properties can be established.
Increasing the distance between the measuring section and the sorting unit often does not offer any practical solution to the problem as an increased distance also increases the risk of beads getting stuck to the wall sections, or getting stuck together, and a longer flow section furthermore contributes to variations in the velocity of individual beads—both of which factors will result in a reduced validity of the results required for a correct sorting of beads.
A further problem often encountered in purely fluid dynamic based bead manipulation methods is that the flow of beads cannot be interrupted on demand without such an interruption causing a considerable shutdown period followed by often laborious and time-consuming start up procedures.
During the operation of a purely fluid based bead sorter, such as e.g. the COPAS bead sorter supplied by Harvard Bioscience, a number of difficulties regarding fluid handling of particles such as e.g. polymer beads are experienced. For example, beads tend to get stuck in the feed tube to the flow cell where the beads are analysed, and two or more beads may therefore stick together as they enter the flow cell. As a result a considerable fraction of beads are not sorted correctly, such as e.g. more than 20%, and the resulting fractions of beads must be resorted to obtain an acceptable level of accuracy. This is time-consuming and inefficient as it requires beads to be measured and analysed more than once.
In order for the beads to line up one by one in the feed tube to the flow cell a high flow velocity of about 1 m/s is required corresponding to a measuring time of less than 1 millisecond per bead and a computation time of less than 100 milliseconds from bead measurement to bead sorting. This places very high demands on sensors, data handling equipment, and the fluid handling components of the sorting unit. In cases where beads are to be imaged even lower exposure times, e.g. 10 microseconds, are required for freezing the motion of a passing bead and obtaining a sharp image.
In cases where advanced bead analysis, such as resolving the three dimensional structure of spatially encoded microparticles must be carried out at a high through-put rate, high flow velocities and corresponding low residence times in the flow cell poses an even greater challenge for the equipment as the data handling is much more complex compared to conventional bead sorters in which a more simple analysis, such as bead size or total fluorescence, is measured.
There is a need for an improved apparatus for bead analysis and sorting which allows high through-put rates while maintaining sufficiently high residence times, e.g. in a compartment or section in which bead properties can be measured, thus ensuring that more reliable data are generated for each and every bead, wherein each bead is preferably only measured once prior to being sorted on the basis of the result and analysis of the measurement of a bead property.
WO 01/77391 A1 (Quantum Dot) discloses devices, systems, kits, and methods for detecting and/or identifying a plurality of spectrally labeled bodies well-suited for performing multiplexed assays. By spectrally labeling the beads with materials which generate identifiable spectra, a plurality of beads may be identified within the fluid. Reading of the beads is facilitated by restraining the beads in arrays, and/or using a focused laser. The present invention is in one aspect directed to a bead sorter comprising a rotatable, circular capture body comprising a plurality of through-going inlets.
WO 99/42209 (Takeda) discloses a bead sorter comprising a bead holder. The bead holder has a surface having a recess sized to receive a single bead, a passage connected at one end thereof to the recess, and a restricting portion for preventing the bead from being entering into the passage. Also, the bead separator has first, second, and third stations. The first station is to introduce a negative pressure in the passage, thereby holding the bead in the recess. The second station is to eject a liquid around the recess retaining the bead, thereby removing a bead or beads possibly existing around the recess away from the recess. The third station is to introduce a positive pressure in the passage, thereby releasing the bead from the recess. A transporting means is provided for moving the recess through the first, second, and then third stations. One embodiment of the present invention is directed to a bead sorter which does not comprise a bead holder having a recess in at least one surface. Rather, the present invention employs in one embodiment a planar disc comprising a plurality of through-going inlets.